The present invention relates to a method of separating blood components. More specifically it relates to a method of producing and obtaining anaphylatoxin- and cocytotaxin-containing leucotaxine preparations and of anaphylatoxin and cocytotaxin proteins in molecularly homogeneous, biologically active form.
Destruction of tissue in inflammation induced by non-immunological and/or immunological processes leads to formation of a variety of endogenous substances (mediators and hormones). They regulate the complex individual steps of activation of inflammation and tissue repair processes. The mediators are produced either as humoral mediators by limited and regulated proteolysis of plasma or serum proteins, or they are released as cellular mediators by active secretion and/or cell lysis from cells and tissues. They form part of the body's defensive system, which systemic and local activation they play part. They thus contribute to removal and detoxification of destroyed endogenous substances and/or invaded foreign bodies. In addition, by regulation of the cell-division and tissue growth processes in wound healing, they participate in restoration of the physiological structures and functions of the organism. Like the classical hormones of the endocrinal glands, inflammatory mediators are trace substances, that are present in situ in only minute concentrations in tissue or blood.
By activation of the kinin system, the coagulation system, the complement system, and also of other blood-protein and cell factors, a variety of mediators may be produced concurrently or sequentially which are responsible for the apparent biological activities of activated serum. Amongst them to mention are chemical attraction of leucocytes (leucotaxis), immunoadherence and the smooth muscle concentrtion. Anaphylatoxin and cocytotaxin are among the blood protein mediators formed by limited, regulated proteolysis of plasma and serum factors in concurrence with complement system activation. They play a major role in the chemical attraction of leucocytes. In addition, anaphylatoxin also possesses pharmacological properties and cardiovascular effects on account of its spasmogenic effects on muscle cells.
Anaphylatoxin was discovered after treating mammalian sera with antigen-antibody complexes; cf. E. Friedberger, Z Immunitatsforsch. 4 (1919), p. 636-689. It is considered as one of the fragments (C5a) of the fifth complement component, with which it has many biological activities in common; cf. J. Jensen, Science 155 (1967), p. 1122-1123. There is still no proof of their chemical identity. Formation of anaphylatoxin and other mediators can be induced by contact reactions of mammalian blood, plasma, or serum with various hydrophilic, insoluble high-molecular substances, such as dextran, yeast, and also with bacterial endotoxins (lipopolysaccharides). Application of such modified sera in vivo and in vitro induces various types of biological effects. Amongst them to mention are the typical anaphylatoxin effects and other reactions which are similar to, or comparable with in vivo immune and non-immune processes apparent in allergy and tissue damage reactions. These biological reactions in particular include release of histamine, lethal shock, contraction of the smooth muscles, and chemotactic activity for neutrophil and eosinophil leucocytes.
A process of ten steps for obtaining anaphylatoxin from rat, pig and guinea-pig serum treated with dextran, yeast or immune complexes, has been described by J. H. Wissler in Eur. J. Immunol. 2(1972), pp. 73--96 and in Int. Arch. Allergy 32 (1972), pp. 722-747. According to this known process, anaphylatoxin is separated from accompanying foreign proteins by chromatography on hydroxyapatite. Prior to this step, an anaphylatoxin-containing crude protein fraction of contact-activiated serum is separated off by cation exchange reaction from the supernatant, negatively absorbed serum components. The anaphylatoxin-containing protein eluate obtained by the cation exchange reaction is concentrated by salting-out precipitation of proteins with ammonium sulfate (serum protein concentrate fraction). Prior to chromatography on hydroxyapatite part of the foreign substances, including a small fraction of accompanying foreign proteins, are removed by treatment with calcium phosphate gel. Following chromatography on hydroxyapatite further purification of anaphylatoxin in carried out in this known process by molecular sieve chromatography, ion-exchange chromatography on hydroxyapatite, and gel-permeation chromatography. Anaphylatoxin is finally obtained in crystalline form in a molecularly homogenous, biologically active state.
By the above process, the anaphylatoxin-containing protein preparation obtained by chromatography on hydroxyapatite, which is chemotactically active for neutrophil leucocytes, is separated into two main protein components. The one protein thus obtained has the classical properties of anaphylatoxin. For example, in vivo it causes release of histamine and it induces the typical lethal anaphylatoxin shock by contraction of the smooth muscles. This substance (classical anaphylatoxin) which is obtained in crystalline form by the known process, however has no appreciable chemotactic activity on neutrophilic leucocytes. This activity is formed by the activation process in crude serum in concurrence with anaphylatoxin activity, as are other activities.
The second protein, termed cocytotaxin, is likewise obtained by the known process in a crystalline state; cf. J. H. Wissler, Eur. J. Immunol. 2 (1972), pp. 84-89. The physicochemical properties of cocytotaxin are similar to anaphylatoxin. But biologically, it is different. Cocytotaxin has no spasmogenic activity of anaphylatoxin. However, as anaphylatoxin, cocytotaxin has no appreciable chemotactic activity on neutrophilic leucocytes.
Recombination of anaphylatoxin and cocytotaxin in various molar ratios in vitro leads to a restoration of the chemotactic activity for neutrophil leucocytes intrinsic to contact-activated crude serum. Thus, anaphylatoxin and cocytotaxin together constitute a leucotaxine preparation as a binary protein system which is biologically active in vitro and in vivo. In this system anaphylatoxin is the activity principle and cocytotaxin constitutes the activity-inducing cofactor (cochemotaxin). Both separated proteins, anaphylatoxin and cocytotaxin, and the leucotaxine preparation containing the two substances as a biologically active binary protein system are valuable substances with a wide array of application possibilities in pathology and immunology. For example, they may be used to induce desired focal inflammation processes, e.g. in tumours ("biochemical surgery"). Accumulating leucocytes can be regulated to be composited of selected cell patterns. In addition, by accumulating selective patterns of leucocyte types they can serve for formation, in statu nascendi, of substances with cell-specific action promoting and inhibiting cell division produced by leucocytes at the reaction site of inflammation. Furthermore, they can be used to increase, in statu nascendi, the immune status at a reaction site in tissue, or tumour sites by immunopotentiators secreted by attracted and accumulating leucocyte populations.
In general, purification processes for proteins and other natural substances consist of a sequence of combined separation techniques. Subtle differences in molecular size, charge, form, structure, stability, and nature of the molecule surface between the desired natural substance and the accompanying foreign substances are used in such steps for separation. Accordingly, a large number of combinations of various separation techniques can be worked out for purification of a protein. The nature and the conditions of separation steps used, but as well thier sequential combination, are of paramount significance for operational properties, technical practicability, optional automatisation possibility and economics of a purification process and for yield and molecular quality of the natural product investigated. Particular attention has to be focused on optimum form of separation steps and on their ingenious combination into a purification sequence within a frame of structural and functional stability and other molecular parameters of the substance under investigation. This implies that use of identical or similar separation principles (e.g. molecular sieve filtration, dialysis ion-exchange absorption, etc.) in a different combination can be of decisive and paramount importance for practice and economics of a purification process and yield and quality of the product obtained. In some cases, use or omission of a single technique (e.g. hydroxyapatite chromatography, zone-precipitation chromatography, etc.) at a certain position in the purification sequence, or within a partial sequence, is decisively significant for yield and quality of the desired natural product as well as for practice and economics of the purification process. These general relationships and basic principles inherent to purification processes of natural products are clearly illustrated e.g. by some well known facts. Thus, within an economically and technically operable process for the purification of a natural product, initial dialysis or lyophilizaton steps are not recommended prior to reduction of initial volumes of the crude extract by a factor ofat least 500-1000 through other techniques.
As above-mentioned, mediators as anaphylatoxin and cocytotaxin are trace substances occurring only in very small quantities in the prepared serum. 100 liters of contact-activated serum (corresponding to 250-300 liters of blood) contain about 7 to 8 kg of protein in addition to other substances. Only about 0,3 to 1,5 g of this protein mass (depending on the species and on the nature of the contact reaction) represent anaphylatoxin, cocytotaxin being present in about twice to three times this quantity. Of this, a maximum of 10 to 20% of each protein can be isolated, since any purification is a complex process having only restricted yields. Therefore, a prerequisite for the practical use of these proteins is their isolation in appreciable quantities. Hence, very large volumes of blood have to be processed.
The above-mentioned process is not suitable for this purpose. Use of hydroxyapatite for structure-conserving purification of these proteins is decisively important. For technical and economic reasons, however, it is not possible to chromatography large volumes of protein solutions on hydroxyapatite columns. Larger amounts of proteins tend to support the strong tendency of hydroxyapatite to block up. Furthermore hydroxyapatite is very expensive. Its use on large scales is not economical. Separation of a part of accompanying foreign proteins achieved in the known process by absorption on calcium phosphate gel admittedly causes a reduction by about 10% of the amount of foreign substance. This reduction is too small to be of practical importance in large scale preparation of these mediators. Thus, the residual volume of protein solution to be applied on the hydroxyapatite column is still too large for economical processing of larger quantities of blood. According to the known process, under optimum conditions, a volume reduction of 1000 ml serum to a minimum of some 25 ml of protein solution can be achieved prior to its application onto the hydroxyapatite column.
Another process for preparation of anaphylatoxin has been described by M. Lieflander et al. in Hoppe-Seyler's Z. Physiol. Chem. 353 (1972), pp. 385-392. Anaphylatoxin is separated from the foreign proteins by repeated cation-exchange chromatography and molecular sieve filtration combined with lyophilization and dialysis steps. Some of the steps are performed in organic solvents and in unbuffered acid solution. E. H. Vallota and H. J. Muller-Eberhardt described a modification of this technique in J. Exp. Med. 137 (1973), pp. 1109-1123, as did H. N. Fernadez et al. in J. Immunol. 120 (1978), pp. 109-115, in which a further cation-exchange chromatographic step and an additional anion-exchange chromatographic step are included. Finally, in J. Biol. Chem. 251 (1979), pp. 6346-6351, C. Gerald and T. E. Hugli suggested another modification of this process for isolation of anaphylatoxin, in which the activated crude serum was first adjusted to pH 0 (1 mol/1 HCl). Without experimental proof, it is implied that these unphysiological conditions do jeopardize the natural biological quality of products investigated. They claimed that 65% of the foreign proteins are precipitated (no experimental data are given). The residual protein fraction is purified further by a molecular sieve filtration, cation-exchange positive absorption chromatography, and anion-exchange negative absorption chromatography.
The above-mentioned known processes are also unsuitable for economical isolation of anaphylatoxin in such quantities as are needed for practical purposes. The use of large volumes of serum would demand too large and too bulky column capacities and almost extremly awkward dialysis and lyophilization steps. In fact, these processes too were conceived and published only for use of relatively small initial serum volumes (from 0.5 to a maximum of 10 liters of contact-activated serum). Moreover, these processes have the disadvantage that irreversible damage of protein structure as a result of the drastic, non-physiological conditions (organic, non-aqueous solvents; pH 0) cannot be excluded or are not investigated, also such events are most likely; see for example J. H. Wissler in Proc. Immunosymposium, Vienna 1973, Springer Verlag Vienna 1975, pp. 91-105. The isolation of cocytotaxin and the separation of residual trace contaminants of other foreign proteins which electrophoretically are hardly detectable, but which are chromatographically obvious, are not envisaged in this method; although their presence is most likely; cf. H. N. Fernandez at al. (op. cit.) and M. C. Conroy et al. in J. Immunol 116 (1976), pp. 1682-1687.
It is therefore, a primary object of this invention to provide a process for producing and obtaining anaphylatoxin- and cocytotaxin-containing leucotaxine preparations from large amounts of blood.
It is another object of this invention to provide a process for producing and obtaining anaphylatoxin and cocytotaxin proteins in molecularly homegenous form.
It is another object of this invention to provide a process for producing and obtaining anaphylatoxin- and cocytotaxin-containing leucotaxine preparations and anaphylatoxin and cocytotaxin proteins in biologically active form.
It is still another object of this invention to provide a process for producing and obtaining anaphylatoxin-and cocytotaxin-containing leucotaxin preparations and anaphylatoxin and cocytotaxin proteins in appreciable quantities in their native intact structure.
It is still another object of this invention to provide a process for producing and obtaining anaphylatoxin- and cocytotaxin-containing leucotaxine preparations and anaphylatoxin and cocytotaxin proteins in a relatively simple, automatable and economical manner.
These and other objects and advantages of the present invention will be evident from the following description of the invention.